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Molecular & Cellular Proteomics 5:2350-2363, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Using Phage Display to Select Antibodies Recognizing Post-translational Modifications Independently of Sequence Context^*

John W. Kehoe^||||,,¶, Nileena Velappan^¶,||, Monica Walbolt, Jytte Rasmussen, Dave King^**, Jianlong Lou, Kristeene Knopp, Peter Pavlik^||, James D. Marks, Carolyn R. Bertozzi^, and Andrew R. M. Bradbury^||,¶¶

From the ^|||| Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, Department of Chemistry, University of California, Berkeley, California 94720-1460, ^|| Biosciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, ^** Howard Hughes Medical Institute Mass Spectrometry Laboratory, University of California, Berkeley, California 94720, Department of Anesthesia and Pharmaceutical Chemistry, University of California, San Francisco General Hospital, San Francisco, California 94110

Many cellular activities are controlled by post-translational modifications, the study of which is hampered by the lack of specific reagents due in large part to their ubiquitous and non-immunogenic nature. Although antibodies against specifically modified sequences are relatively easy to obtain, it is extremely difficult to derive reagents recognizing post-translational modifications independently of the sequence context surrounding the modification. In this study, we examined the possibility of selecting such antibodies from large phage antibody libraries using sulfotyrosine as a test case. Sulfotyrosine is a post-translational modification important in many extracellular protein-protein interactions, including human immunodeficiency virus infection. After screening almost 8000 selected clones, we were able to isolate a single specific single chain Fv using two different selection strategies, one of which included elution with tyrosine sulfate. This antibody was able to recognize sulfotyrosine independently of its sequence context in test peptides and a number of different natural proteins. Antibody reactivity was lost by antigen treatment with sulfatase or preincubation with soluble tyrosine sulfate, indicating its specificity. The isolation of this antibody signals the potential of phage antibody libraries in the derivation of reagents specific for post-translational modifications, although the extensive screening required indicates that such antibodies are extremely rare.

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